摘要
为研究PepT1 mRNA在鸡小肠的表达对于蛋白质消化吸收的影响,实验建立了PepT1基因绝对荧光定量PCR标准曲线。根据GenBank中PepT1的序列信息设计合成两对引物,引物一是克隆引物,用于从鸡小肠细胞中扩增出cDNA片段;引物二是荧光定量PCR引物,其长度包含于引物一中,用荧光定量PCR来检测基因的表达。通过克隆出PepT1基因282 bp部分片段并插入到PMD18载体中,从而构建出绝对荧光定量PCR标准曲线。从实验结果得出标准曲线的回归方程为Y=-3.205X+34.170,其回归系数R2=0.990,溶解曲线表现为单一的峰,通过对结果的具体分析,认为该方法可靠,特异性均较强,可成功构建目的基因的标准曲线。
To study the effect of PepT1 mRNA expression in chicken intestine on dietary protein absorption,we designed two pairs of primers according the Genbank PepT1 sequence information for quantifing the gene＇s expression using real-time PCR.A 282 bp segment of PepT1 gene was cloned and inserted into the PMD18 vector for creating the standard curve of absolute quantification.A liner regression equation（Y=-3.205X＋34.17） was obtained from our experimental result and the regression coefficient（R2） of the standard curve is 0.990.Referred to the dissociation curve analysis,we consider the method is reliable,specificity and exercisable to generate standard curve of target gene successfully.
出处
《山西农业大学学报（自然科学版）》
CAS
2010年第4期332-335,共4页
Journal of Shanxi Agricultural University(Natural Science Edition)
基金
863项目(2008AA101009)
中国博士后科学基金(20090451360)
山西省回国留学人员科研资助项目(2009035)
山西农业大学科技创新基金(200902)
